(1) Field of the Invention
The field of this invention is nucleic acid chemistry, more specifically nucleic acid amplification technology, and more specifically isothermal amplification methods. This invention relates to processes that, without raising or lowering the temperature, increase the number of copies (“amplify”) of a specific “target” DNA or RNA (collectively xNA) sequence.
(2) Description of Related Art
For practical applications in many areas, including the research and DNA- and RNA-targeted diagnostics, methods that amplify nucleic acids without the need to do temperature cycling are highly desirable. Many such “isothermal amplification” methods are known in the art, including those known as “recombinase polymerase amplification” (RPA) [Piepenburg, O., Williams, C. H., Stemple, D. L., Armes, N. A. (2006) DNA Detection Using Recombination Proteins. PLoS Blot 4 (7): e204. doi:10.1371/journal.pbio.0040204. PMC 1475771. PMID 16756388.], rolling circle amplification (RCA), helicase-dependent amplification (HDA), NASBA, and LAMP, among others
Isothermal amplification methods frequently do not perform well, however. In many cases, the extent of amplification appears to depend on the specific sequence being amplified or (perhaps) the sequence of probes and/or primers used in the amplification. In some cases, the amplification fails entirely. When the amplification targets more than one target nucleic acid species, isothermal amplification methods often fail.
Essentially no theory explains these and other variable results, although speculation can be found in the public and private art, some of it contradictory, other examples being informal. Without any attempt to be exhaustive, speculative suggestions include the possibility that at low temperatures, non-Watson Crick interactions might cause some of the DNA molecules involved (primer, probe, or analyte) to fold in a way that defeats the amplification process. Others have suggested that high temperatures must be regularly traversed to avoid an (often unknown) intra- or intermolecular interaction from capturing the system as an artifact. Primer-primer interactions have been invoked to explain failure of various isothermal amplification systems, especially when is multiplexing is attempted.
None of these explanations are established. Few data allow us to prefer one over the other. As a consequence, the art contains no clear guidance as to what experiments might be tried to overcome these problems, and to generate reliable procedures of performing isothermal amplification for all target sequences and, especially, for multiple (more than one) target sequences.